RESEARCH ARTICLE
Magnetic resonance imaging and spectroscopy for differential assessment of liver abnormalities induced by Opisthorchis felineus in an animal model Alexandra G. Pershina1,2*, Vladimir V. Ivanov1, Lina V. Efimova1, Oleg B. Shevelev3, Sergey V. Vtorushin1, Tatjana V. Perevozchikova1, Alexey E. Sazonov1, Ludmila M. Ogorodova1
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1 Central Research Laboratory, Siberian State Medical University, Tomsk, Russia, 2 Department of Biotechnology and Organic Chemistry, National Research Tomsk Polytechnic University, Tomsk, Russia, 3 Center for Genetic Resources of Laboratory Animals, Institute of Cytology and Genetics SB RAS, Novosibirsk, Russia * [email protected]
OPEN ACCESS Citation: Pershina AG, Ivanov VV, Efimova LV, Shevelev OB, Vtorushin SV, Perevozchikova TV, et al. (2017) Magnetic resonance imaging and spectroscopy for differential assessment of liver abnormalities induced by Opisthorchis felineus in an animal model. PLoS Negl Trop Dis 11(7): e0005778. https://doi.org/10.1371/journal. pntd.0005778 Editor: jong-Yil Chai, Seoul National University College of Medicine, REPUBLIC OF KOREA Received: March 31, 2017
Abstract Background European liver fluke Opisthorchis felineus, causing opisthorchiasis disease, is widespread in Russia, Ukraine, Kazakhstan and sporadically detected in the EU countries. O. felineus infection leads to hepatobiliary pathological changes, cholangitis, fibrosis and, in severe cases, malignant transformation of bile ducts. Due to absence of specific symptoms, the infection is frequently neglected for a long period. The association of opisthorchiasis with almost incurable bile duct cancer and rising international migration of people that increases the risk of the parasitic etiology of liver fibrosis in non-endemic regions determine high demand for development of approaches to opisthorchiasis detection.
Accepted: July 5, 2017 Published: July 14, 2017 Copyright: © 2017 Pershina et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by the Russian Science Foundation (http://www.rscf.ru/en) [grant number 14-15-00247 to AGP, AES, LMO], and the Russian Foundation for Basic Research (http:// www.rfbr.ru/rffi/eng) [grant number 15-04-05580 a to VVI]. Animal MRI/MRS was conducted at the
Methodology/Principal findings In vivo magnetic resonance imaging and spectroscopy (MRI and MRS) were applied for differential assessment of hepatic abnormalities induced by O. felineus in an experimental animal model. Correlations of the MR-findings with the histological data as well as the data of the biochemical analysis of liver tissue were found. MRI provides valuable information about the severity of liver impairments induced by opisthorchiasis. An MR image of O. felineus infected liver has a characteristic pattern that differs from that of closely related liver fluke infections. 1H and 31P MRS in combination with biochemical analysis data showed that O. felineus infection disturbed hepatic metabolism of the host, which was accompanied by cholesterol accumulation in the liver.
Conclusions A non-invasive approach based on the magnetic resonance technique is very advantageous and may be successfully used not only for diagnosing and evaluating liver damage induced
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MRI and MRS of Opisthorchis felineus infected liver
Centre for Genetic Resources of Animal Laboratory at the Institute of Cytology and Genetics, Siberian Branch, Russian Academy of Sciences (Ministry of Education and Science of the Russian Federation (http://www.fcpir.ru/) projects Nos. RFMEFI61914X0005 and RFMEFI62114X0010). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.
by O. felineus, but also for investigating metabolic changes arising in the infected organ. Since damages induced by the liver fluke take place in different liver lobes, MRI has the potential to overcome liver biopsy sampling variability that limits predictive validity of biopsy analysis for staging liver fluke-induced fibrosis.
Author summary Opisthorchiasis caused by Opisthorchis felineus is a fish-borne parasitic worm infection spread in Russia and some European countries. The morbidities provoked by O. felineus infection are cholangitis and bile duct fibrosis. Long-term infection is associated with high risk of developing cholangiocarcinoma, a generally incurable bile duct cancer type. Due to lack of specific symptoms, O. felineus infection often escapes detection. Thus, opisthorchiasis diagnosis, especially in non-endemic regions, is a serious problem for physicians. In the paper, an animal model of O. felineus induced opisthorchiasis has been evaluated by in vivo magnetic resonance spectroscopy (MRS) and magnetic resonance imaging (MRI). Application of MR-techniques allowed to detect a characteristic MRI pattern of O. felineus infected liver, get valuable information about the severity of organ impairments, and bring to light some metabolic changes provoked by the helminth in the liver. Since damages take place in different liver lobes, MRI has the potential to overcome sampling variability of liver biopsy that limits liver fluke-induced fibrosis staging. The use of MRtechniques is very advantageous for investigating parasitic infection. Collection of experimental MR-data gives a new impulse to examination of infected humans and encourages to implement these methods in routine diagnosis of infections, including but not limited to opisthorchiasis.
Introduction Two liver fluke species of the genus Opisthorchis—O. felineus and O. viverrini—are known as human pathogenic agents. This fluke parasite inhabits the bile duct of the host liver, causing local mechanical damage and chronic inflammation, and having general toxic effects on the whole body [1]. Bile duct dilatation and mechanical obstruction accompanied by bile sludge formation are common complications of chronic opisthorchiasis [2]. Generally, liver fluke infection causes hepatobiliary pathological changes and leads to cholangitis, cholecystitis, and cholelithiasis. The most severe complication of opisthorchiasis is malignant transformation. There is a significant association between cholangiocarcinoma (CAA) and O. viverrini infection; that liver fluke is considered carcinogenic (group I) to humans [3–5]. O. felineus is classified by IARC as group 3 due to limited experimental data [6]. However, in sources published in the Russian language, the association between CAA and chronic long-term O. felineus infection has been emphasized [7,8]. In whole, periductal fibrosis is considered an important risk factor for bile duct cancer and is associated with CCA development [9,10]. In the meantime, there is no significant association between liver fluke infection and cirrhosis, at least for closely related species O. viverrini and Clonorchis sininsis [5]. Chronic liver diseases, mainly liver fibrosis, represent a public health problem worldwide [11]. The generally discussed disease entities associated with liver fibrosis are nonalcoholic fatty liver disease (NAFLD), alcoholic liver disease, chronic viral hepatitis and chronic
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MRI and MRS of Opisthorchis felineus infected liver
cholestasis, primary biliary cholangitis, and primary sclerosis cholangitis [12]. Fibrosis induced by liver flukes is commonly out of focus, though chronic infection caused by helminths of the genus Opisthorchis leads to periductal fibrosis and severe abnormalities in the hepatobiliary system [3]. It is clear that the pathogenesis of liver fibrosis depends on the underling etiology [13]. The main reason for complicated recognition of the cause of fibrotic or inflammatory process consists in the fact that liver responds to various injuries in a limited number of ways [14]. Parasitic liver flukes of the genus Opisthorchis are widespread in Eurasia—in Russia (mainly West Siberia), Ukraine, Kazakhstan (O. felineus), and South Asia (O. viverrini). In the endemic regions more than 750 million people are at high risk of liver fluke infection [5]. There are some reports about O. felineus infected people in EU countries [15,16]. It is very important to note that most infected persons, especially those with chronic infection (liver fluke lifespan may exceed 25 years [4]), show no symptoms at all or non-specific symptoms which are very difficult to recognize, so the infection is frequently neglected [17,18]. Taking into consideration increasing international migration of people, it is essential to bear in mind the risk of the parasitic etiology of liver fibrosis in the non-endemic regions [19]. In time, non-detected opisthorchiasis infection may be a cause of false diagnosis and complications in the treatment strategy of choice [17]. Furthermore, it is very important to exclude liver fluke infection, when assessing the transplantation potential of living liver donors. Search for non-invasive tests for liver fibrosis diagnosis is the main trend in hepatology, since the “gold-standard” method—liver biopsy—has some shortcomings, such as invasiveness, complications, and sampling variability [11,13,20]. MR imaging-based techniques for liver fibrosis assessment are very promising and are being actively developed currently [21]. Magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (MRS) share common physical background and are based on a nuclear magnetic resonance (NMR) phenomenon [22]. In the presence of a static magnetic field, for a nucleus of spin 1/2, absorption of energy emitted by a radiofrequency coil induces "flips" of a magnetic moment to another orientation. This "spin flip" places some of the spins in their high energy state. Relaxation processes which return nuclei back to the lower energy state after switching off the radiofrequency signal come with generation of a measurable amount of the radio frequency signal. The produced NMR signals received by the radiofrequency coil allow to generate MRS and MRI after Fourier transformation. MRI has become an increasingly important imaging technique for investigating patients with hepatic and biliary disorders [23,24]. Although MRI examination is more expensive in comparison with computed tomography and ultrasound, this modality is widely used due to higher spatial resolution [25] and absence of ionizing radiation. MRS methods offer an opportunity to assess relative tissue metabolite concentrations based on the chemical shift phenomenon [26] and are very useful in clinical and biomedical studies for examining metabolic changes in vivo non-invasively [25,27]. In particular, 1H MRS is successfully used to determine relative lipid concentration in hepatic tissue [28], whereas 31P MRS allows to detect phosphorylated metabolites, including high-energy phosphates [25]. MRS methods are implemented to investigate the hepatic metabolic state and are applied as non-invasive diagnostic techniques for studying hepatobiliary pathology [29–31]. Despite the fact that MRI and MRS have become increasingly important imaging techniques for investigation of patients with liver and biliary disease [23,24], there are few papers describing application of in vivo MR-techniques for studying liver fluke infection [32–35] and no papers depicting the use of these methods for investigating O. felineus. In this study we used magnetic resonance techniques (MRI/MRS) for differential assessment of liver abnormalities induced by O. felineus in an experimental animal model. We also
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MRI and MRS of Opisthorchis felineus infected liver
found out correlations between the MR-findings and the histological data as well as the data of the biochemical analysis of liver tissue.
Materials and methods Ethics statement The experimental protocol was approved by the Bioethics Review Committee of the Institute of Cytology and Genetics, Siberian Branch, Russian Academy of Sciences, Novosibirsk (No. 30 from 20.11.2015). All animal experiments were conducted according to the principles of the Guide for the care and use of laboratory animals [36].
Experimental opisthorchiasis model Metacercariae of O. felineus were obtained from naturally infected fish (Leuciscus leuciscus) caught in the river in the endemic areas of Western Siberia (the Tom, Tomsk), Russia. A permit was not required to collect these fish according Federal law of the Russian Federation No. 166 “About fisheries and conservation of water biological resources” from 20.12.2004. For the experiment, 5-week-old male hamsters (n = 8) (Mesocricetus auratus) were infected intragastrically with 50 metacercariae per hamster, according to the previously described protocol [37]. Age-matched intact male hamsters (n = 8) were used as controls. The hamsters were housed two in a cage (OptiRAT) under conventional conditions and were permitted ad libitum access to food and water. The animals were handled in pathogen-free environment. At 8 weeks post-infection, the hamsters were scanned with MRI and MRS in vivo. Following the MR examinations, the subsets of the infected and intact animals were deeply anesthetized with carbon dioxide and euthanized by decapitation. Blood and liver samples were collected for examination from each hamster from the control (n = 8) and infected (n = 8) groups. Serum and blood analyses were performed using routine procedures (Methods in S1 Text).
MRI and MRS in vivo All 1H/31P MR experiments were performed on a horizontal tomographic scanner with magnetic field intensity of 11.7 T (Bruker, Biospec 117/16 USR, Germany). Prior to MR examinations, the animals were fasted overnight. The animals were anaesthetized with gas anesthesia (Isofluran; Baxter Healthcare Corp., Deerfield, IL) using a Univentor 400 Anesthesia Unit (Univentor, Zejtun, Malta). The animal body temperature was maintained with a water circuit installed into the table bed of the tomographic scanner, which maintained the temperature of 30˚C on its surface. A pneumatic respiration sensor (SA Instruments, Stony Brook, NY) was placed under the lower body part, which allowed to control the anesthesia depth. MRI. All MR images were recorded with a receiver—transmitter 1H volume coil (T11440V3). High-resolution T2-weighted images of the hamster liver (section thickness 1 mm; field of vision (FOV) 4.0 × 4.0 mm; matrix 512 × 512 dots) were acquired with respiratory triggering using TurboRARE (Rapid Imaging with Refocused Echoes) method with pulse sequence parameters: TEeff = 18 ms, TR = 900 ms, Flip Angle = 180˚, RARE Factor = 4. Serial images in axial and coronal orientation were recorded for each animal. 1H/31P-MRS. Proton and phosphorus spectra were recorded with a receiver—transmitter double-tuned 1H/31P surface coil (T11619V3). The coil was positioned to the right upper abdominal quadrant of the hamster liver in the right lateral decubitus position, which allowed to minimize the signal capture by muscles and decrease movement artifacts. Before each session, recording of 3 orthogonal packages of liver sections (Multi-slice Tri-Pilot scanning) was conducted, to guide the correct coil positioning relative to liver tissue (in case of infected
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MRI and MRS of Opisthorchis felineus infected liver
hamster to capture liver damaged zone). The protons spectra were recorded using the spatially localized PRESS (Point Resolved Spectroscopy) method with TE = 20 ms, TR = 2.5 ms, and 256 replicates, voxel size 5×5×5 mm. Water signal in the spectra was suppressed by the variable power and optimized relaxation delays (VAPOR) method. The phosphorus spectra were recorded using the non-spatially localized single-pulse method with TE = 3 ms, TR = 100 ms, and 2,048 replicates. The spectrum range was 40 ppm, which made it possible to obtain information about the tissue contents of 5 substances represented by 7 peaks, phosphomonoester (PME; 6 ppm), inorganic phosphate (Pi; 5 ppm), phosphodiester (PDE; 2 ppm), creatine phosphate (PCr; 0 ppm originated from muscle contamination), and nucleoside triphosphate (NTP; 2.7, 7.8, and 16.5 ppm). MRS data processing. Time domain data were Fourier transformed after Gaussian multiplication (center, 0 ms; width, 30 ms) and phase corrected. Quantification of the spectral peak areas was performed using the TOPSPIN 2.0 PV software package (Brucker), including polynomial baseline correction followed by frequency domain curve peak integration. The 1H and 31P metabolite concentrations were calculated from the peak areas and expressed relative to an area under the curve. Intracellular pH was calculated based on the chemical shift difference δ between Pi and α-NTP (δ = fPi-fαNTP-7.56) using the Henderson-Hasselbalch equation [38]: pHi ¼ 6:75 þ log10½ðdPi
3:27Þ=ð5:69
dPiÞ
ð1Þ
Histological analysis Liver samples (from posterior segments of the right lobe) were fixed in 10% buffered formalin, and embedded in paraffin. Tissue sections were cut into 4–5 μm-thick slices and stained with hematoxylin and eosin. Histological analysis was performed with the optical microscope Axiostar plus (Carl Zeiss, Germany). For differential staining of collagen in the liver samples, Van Gieson’s method was used. Fibrosis was graded according to METAVIR score [39]. Hepatic steatosis was graded on a 0–3 scale through visual estimation of the percentage of hepatocytes containing intracellular vacuoles of fat [40].
Liver tissue analysis Two samples of liver tissue were taken from posterior segments of the right lobe and medial segments of the left lobe for each animal. The samples were divided into five pieces in accordance with subsequent examinations and immediately placed in liquid nitrogen. Homogenization was performed using Tissue Grinders (PELLET PESTLE1 Cordless Motor, Kimble Chase, TN) on ice. Lipids assay. Extraction of lipids from 200 mg of liver tissue (two pieces for each animal) was performed using Folch method [41]. The concentrations of cholesterol, triglycerides and phospholipids in the lipid extract were determined spectrophotometrically (SF2000, Russia) using Chronolab kits (Spain). The total lipids were gravimetrically determined by drying 2 ml of liver lipid extract in a glass vial. ATP assay. 60 mg of liver tissue (two pieces for each animal) was homogenized in 1 ml of 3% trichloroacetic acid and centrifuged at 4,000 g, 4˚C, 20 min. The pH of the supernatant was adjusted to 7.4 with 1 M Tris. The ATP concentration was measured on Anthos lucy2 (Asys Hitech GmbH, Austria) using the Adenosine 50 -triphosphate (ATP) Bioluminescent Assay Kit (Sigma-Aldrich, St. Louis, MO). Protein assay. 30 mg of liver tissue (two pieces for each animal) was homogenized in 400 μl of NP-40 buffer (150 mM sodium chloride, 1.0% Triton X-100, 50 mM Tris, pH 8.0) and centrifuged at 12,000 g, 4˚C, 20 min. Then the protein concentration was determined in
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the supernatant using Bradford assay [42]. Bovine serum albumin (BSA fraction V, Sigma) was used as a protein standard. Glycogen assay. 20 mg of liver tissue (two pieces for each animal) was homogenized in 1 ml of 0.3 M HClO4. The homogenate was incubated with aminoglucosidase (AG, Sigma) in 50 mM sodium acetate, 0.02% BSA, pH 5.5 for 2 h at RT, and centrifuged at 10,000 g for 1 min. The amount of the released glucose was determined by the Hexokinase Method [43] using the Glucose Reagent (Vector-Best, Russia). Glucose (Sigma) was used as a standard. Free glucose in the homogenate was measured without addition of AG and subtracted from AG-treated samples. Western blot. 20 mg of liver tissue was homogenized on ice using dounce homogenizer in 1 ml of lysis buffer (50 mM Tris-HCl (pH 7.5), 250 mM sucrose, 5 mM sodium pyrophosphate (NaPPi), 50 mM NaF, 1 mM EDTA, 1 mM dithiothreitol (DTT), 0.5 mM phenylmethylsulfonyl fluoride (PMSF)) supplemented with Mammalian Protease inhibitor Cocktail (P8340 Sigma). The protein concentration was quantified by Bradford assay, using BSA (Sigma) as a standard. Following lysis sodium dodecyl sulfate (SDS) was added to a final concentration of 0.2% and samples are boiled for 5 min. The samples (twenty micrograms of total proteins) were loaded onto a 15% SDS-PAGE gel according to Laemmli protocol [44]. The separated proteins were transferred to nitrocellulose membranes. The membranes were blocked with 3% non-fat milk in Tris-buffered saline (TBS) with 0.1% Tween 20 and 50 mM NaF for 1 h at room temperature and probed overnight at 4˚C with primary antibodies in 5% BSA supplemented with 50 mM NaF, followed by incubation with Goat Anti-Rabbit IgG H&L (HRP) (ab6721) (Abcam, USA) in TBS supplemented with 5% milk and 50 mM NaF for 1 h at room temperature. The membranes were developed with a ECL Chemiluminescent Substrate Reagent Kit (Thermo Scientific, USA) and scanned by G:box XT4 (Syngene). The band intensities were quantified using GeneTools (Synegene). The primary antibodies Phospho-AMPKα (Thr172) (CellSignaling technology, Inc.) and Anti-beta Actin antibody (ab8227) (Abcam, USA) were used.
Statistical analysis Statistical analyses were performed using IBM SPSS Statistics for Windows, Version 21.0 (Armonk, NY: IBM Corp.). Data were presented as median (range) for data with non-normal distribution and mean (SD) for data with normal distribution. The data were tested by the Shapiro—Wilk test for normality. The differences among continuous variables with normal distribution were analyzed by the t-test and among continuous variables with non-normal distribution—by the Mann-Whitney test. For correlation analyses, the Person (for continuous variables) and Spearman (for ordinal variables) correlations were used. The P value below 0.05 was considered as significant.
Results Histological analysis Histological analysis data showed that O. felineus infection resulted in derangement of the liver architecture, whereas the trabecular pattern mostly remained intact. Hepatocytes had various sizes and patchy exhibited cloudy swelling. Cholangitis in the portal tract was accompanied by pronounced periductal and mild portal fibrosis as well as focal cystic dilatations of the intrahepatic bile ducts. Small bile ductular proliferation and irregular periportal (mostly moderate) infiltration of inflammatory cells, with predominant population of lymphocytes and histiocytes, occurred (Fig 1). Fibrosis was irregular; patchy, incomplete, thin fibrous septa (portal to portal and portal to central bridge) were observed (Fig 1A, 1B and 1C). In whole, chronic
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MRI and MRS of Opisthorchis felineus infected liver
Fig 1. Histological analysis of hamster liver. (A) Microphotographs of Opisthorchis felineus infected hamster liver at 8 weeks post-infection. An adult fluke is found in bile duct lumen. Small bile duct proliferation and chronic cholangitis accompanied by inflammatory infiltration in the bile duct wall; hematoxylin-eosin staining, ×100. (B) Mild portal fibrosis with thin septa formation (portal to portal and portal to central) and (C) pronounced periductal fibrosis with chronic inflammation around the bile duct are observed. In adjacent hepatic tissue, fibrosis with porto-portal septa formation takes place. Van Gieson’s staining showing extensive deposition of mature collagen fibers, ×100. (D) Microphotographs of the uninfected hamster liver. No pathological changes are observed. Hematoxylin-eosin staining, ×100 and (E) Van Gieson’s staining, ×100. https://doi.org/10.1371/journal.pntd.0005778.g001
cholangitis (mild to moderate inflammatory activity, A1-A2 according to METAVIR score) and mild chronic hepatitis were diagnosed, and the stage of fibrosis varied from 1 to 2 according to METAVIR score. There were no lipid droplets in the tested liver samples. No pathological changes took place in the liver in the reference group (Fig 1D and 1E).
Blood and serum analysis The results of blood and serum analyses are given in Results in S1 Text and Table in S1 Table. In the infected group of hamsters the levels of alanine aminotransferase (ALT) and gammaglutamyl transferase (GGT) increased markedly. Significant elevation of cholesterol, triglycerides, and low-density lipoproteins (LDL) in serum of the infected animals was detected. All the above listed serum parameters correlated with the fibrosis stage. The concentration of albumin in serum of the infected hamsters was lower than in the reference group and was accompanied by a statistically significant increase in the urea concentration. However, there was no correlation between these parameters (r = -0.271, p = 0.309).
Biochemical analysis of liver tissue The liver to body index increased in the infected animals, while the spleen to body index remained unchanged (S1 Table). The results of liver tissue biochemical analysis are given in Table 1. There was a statistically significant rise in the cholesterol level in opisthorchiasis, and the cholesterol to phospholipid ratio also elevated in the infected liver. Total lipid content, triglycerides and phospholipids as well as glycogen did not differ between the control and infected groups of hamsters. A significant decrease in the protein concentration occurred in the infected livers. However, the calculated total protein content in the entire liver did not differ between both control and experimental groups due to enlargement of the organ in the infected hamsters. It is important to note, that infection did not lead to lowering of the ATP concentration in liver tissue.
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MRI and MRS of Opisthorchis felineus infected liver
Table 1. Liver tissue biochemical analysis. Metabolite#
Control
Protein, mg g-1 Total cholesterol, mg g
-1
Infected
Correlation with fibrosis stage
mean
SD
mean
SD
r
p-level
120.80
17.19
94.92*
15.62
-0.720
0.002
1.80
0.25
2.64*
0.59
0.781
0.000
Triglycerides, mg g-1
2.84
0.39
3.29
1.23
0.282
0.290
Phospholipids, mg g-1
9.89
0.84
8.93
1.05
-0.447
0.082
Total lipids, mg g-1
42.77
4.91
39.88
4.38
-0.136
0.615
Triglycerides to phospholipid ratio
0.26
0.04
0.32
0.09
0.463
0.071
Cholesterol to phospholipid ratio
0.37
0.06
0.60*
0.10
0.885
0.000
ATP, nmol g-1
2.77
0.66
2.94
1.04
-0.032
0.905
Glycogen, mg g-1
4.17
3.41
6.11
2.16
0.152
0.573
#
Concentration calculated per gram wet weight of tissue
*difference between the infected and the control group is significant at p
Magnetic resonance imaging and spectroscopy for differential assessment of liver abnormalities induced by Opisthorchis felineus in an animal model Alexandra G. Pershina1,2*, Vladimir V. Ivanov1, Lina V. Efimova1, Oleg B. Shevelev3, Sergey V. Vtorushin1, Tatjana V. Perevozchikova1, Alexey E. Sazonov1, Ludmila M. Ogorodova1
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1 Central Research Laboratory, Siberian State Medical University, Tomsk, Russia, 2 Department of Biotechnology and Organic Chemistry, National Research Tomsk Polytechnic University, Tomsk, Russia, 3 Center for Genetic Resources of Laboratory Animals, Institute of Cytology and Genetics SB RAS, Novosibirsk, Russia * [email protected]
OPEN ACCESS Citation: Pershina AG, Ivanov VV, Efimova LV, Shevelev OB, Vtorushin SV, Perevozchikova TV, et al. (2017) Magnetic resonance imaging and spectroscopy for differential assessment of liver abnormalities induced by Opisthorchis felineus in an animal model. PLoS Negl Trop Dis 11(7): e0005778. https://doi.org/10.1371/journal. pntd.0005778 Editor: jong-Yil Chai, Seoul National University College of Medicine, REPUBLIC OF KOREA Received: March 31, 2017
Abstract Background European liver fluke Opisthorchis felineus, causing opisthorchiasis disease, is widespread in Russia, Ukraine, Kazakhstan and sporadically detected in the EU countries. O. felineus infection leads to hepatobiliary pathological changes, cholangitis, fibrosis and, in severe cases, malignant transformation of bile ducts. Due to absence of specific symptoms, the infection is frequently neglected for a long period. The association of opisthorchiasis with almost incurable bile duct cancer and rising international migration of people that increases the risk of the parasitic etiology of liver fibrosis in non-endemic regions determine high demand for development of approaches to opisthorchiasis detection.
Accepted: July 5, 2017 Published: July 14, 2017 Copyright: © 2017 Pershina et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by the Russian Science Foundation (http://www.rscf.ru/en) [grant number 14-15-00247 to AGP, AES, LMO], and the Russian Foundation for Basic Research (http:// www.rfbr.ru/rffi/eng) [grant number 15-04-05580 a to VVI]. Animal MRI/MRS was conducted at the
Methodology/Principal findings In vivo magnetic resonance imaging and spectroscopy (MRI and MRS) were applied for differential assessment of hepatic abnormalities induced by O. felineus in an experimental animal model. Correlations of the MR-findings with the histological data as well as the data of the biochemical analysis of liver tissue were found. MRI provides valuable information about the severity of liver impairments induced by opisthorchiasis. An MR image of O. felineus infected liver has a characteristic pattern that differs from that of closely related liver fluke infections. 1H and 31P MRS in combination with biochemical analysis data showed that O. felineus infection disturbed hepatic metabolism of the host, which was accompanied by cholesterol accumulation in the liver.
Conclusions A non-invasive approach based on the magnetic resonance technique is very advantageous and may be successfully used not only for diagnosing and evaluating liver damage induced
PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0005778 July 14, 2017
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MRI and MRS of Opisthorchis felineus infected liver
Centre for Genetic Resources of Animal Laboratory at the Institute of Cytology and Genetics, Siberian Branch, Russian Academy of Sciences (Ministry of Education and Science of the Russian Federation (http://www.fcpir.ru/) projects Nos. RFMEFI61914X0005 and RFMEFI62114X0010). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.
by O. felineus, but also for investigating metabolic changes arising in the infected organ. Since damages induced by the liver fluke take place in different liver lobes, MRI has the potential to overcome liver biopsy sampling variability that limits predictive validity of biopsy analysis for staging liver fluke-induced fibrosis.
Author summary Opisthorchiasis caused by Opisthorchis felineus is a fish-borne parasitic worm infection spread in Russia and some European countries. The morbidities provoked by O. felineus infection are cholangitis and bile duct fibrosis. Long-term infection is associated with high risk of developing cholangiocarcinoma, a generally incurable bile duct cancer type. Due to lack of specific symptoms, O. felineus infection often escapes detection. Thus, opisthorchiasis diagnosis, especially in non-endemic regions, is a serious problem for physicians. In the paper, an animal model of O. felineus induced opisthorchiasis has been evaluated by in vivo magnetic resonance spectroscopy (MRS) and magnetic resonance imaging (MRI). Application of MR-techniques allowed to detect a characteristic MRI pattern of O. felineus infected liver, get valuable information about the severity of organ impairments, and bring to light some metabolic changes provoked by the helminth in the liver. Since damages take place in different liver lobes, MRI has the potential to overcome sampling variability of liver biopsy that limits liver fluke-induced fibrosis staging. The use of MRtechniques is very advantageous for investigating parasitic infection. Collection of experimental MR-data gives a new impulse to examination of infected humans and encourages to implement these methods in routine diagnosis of infections, including but not limited to opisthorchiasis.
Introduction Two liver fluke species of the genus Opisthorchis—O. felineus and O. viverrini—are known as human pathogenic agents. This fluke parasite inhabits the bile duct of the host liver, causing local mechanical damage and chronic inflammation, and having general toxic effects on the whole body [1]. Bile duct dilatation and mechanical obstruction accompanied by bile sludge formation are common complications of chronic opisthorchiasis [2]. Generally, liver fluke infection causes hepatobiliary pathological changes and leads to cholangitis, cholecystitis, and cholelithiasis. The most severe complication of opisthorchiasis is malignant transformation. There is a significant association between cholangiocarcinoma (CAA) and O. viverrini infection; that liver fluke is considered carcinogenic (group I) to humans [3–5]. O. felineus is classified by IARC as group 3 due to limited experimental data [6]. However, in sources published in the Russian language, the association between CAA and chronic long-term O. felineus infection has been emphasized [7,8]. In whole, periductal fibrosis is considered an important risk factor for bile duct cancer and is associated with CCA development [9,10]. In the meantime, there is no significant association between liver fluke infection and cirrhosis, at least for closely related species O. viverrini and Clonorchis sininsis [5]. Chronic liver diseases, mainly liver fibrosis, represent a public health problem worldwide [11]. The generally discussed disease entities associated with liver fibrosis are nonalcoholic fatty liver disease (NAFLD), alcoholic liver disease, chronic viral hepatitis and chronic
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cholestasis, primary biliary cholangitis, and primary sclerosis cholangitis [12]. Fibrosis induced by liver flukes is commonly out of focus, though chronic infection caused by helminths of the genus Opisthorchis leads to periductal fibrosis and severe abnormalities in the hepatobiliary system [3]. It is clear that the pathogenesis of liver fibrosis depends on the underling etiology [13]. The main reason for complicated recognition of the cause of fibrotic or inflammatory process consists in the fact that liver responds to various injuries in a limited number of ways [14]. Parasitic liver flukes of the genus Opisthorchis are widespread in Eurasia—in Russia (mainly West Siberia), Ukraine, Kazakhstan (O. felineus), and South Asia (O. viverrini). In the endemic regions more than 750 million people are at high risk of liver fluke infection [5]. There are some reports about O. felineus infected people in EU countries [15,16]. It is very important to note that most infected persons, especially those with chronic infection (liver fluke lifespan may exceed 25 years [4]), show no symptoms at all or non-specific symptoms which are very difficult to recognize, so the infection is frequently neglected [17,18]. Taking into consideration increasing international migration of people, it is essential to bear in mind the risk of the parasitic etiology of liver fibrosis in the non-endemic regions [19]. In time, non-detected opisthorchiasis infection may be a cause of false diagnosis and complications in the treatment strategy of choice [17]. Furthermore, it is very important to exclude liver fluke infection, when assessing the transplantation potential of living liver donors. Search for non-invasive tests for liver fibrosis diagnosis is the main trend in hepatology, since the “gold-standard” method—liver biopsy—has some shortcomings, such as invasiveness, complications, and sampling variability [11,13,20]. MR imaging-based techniques for liver fibrosis assessment are very promising and are being actively developed currently [21]. Magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (MRS) share common physical background and are based on a nuclear magnetic resonance (NMR) phenomenon [22]. In the presence of a static magnetic field, for a nucleus of spin 1/2, absorption of energy emitted by a radiofrequency coil induces "flips" of a magnetic moment to another orientation. This "spin flip" places some of the spins in their high energy state. Relaxation processes which return nuclei back to the lower energy state after switching off the radiofrequency signal come with generation of a measurable amount of the radio frequency signal. The produced NMR signals received by the radiofrequency coil allow to generate MRS and MRI after Fourier transformation. MRI has become an increasingly important imaging technique for investigating patients with hepatic and biliary disorders [23,24]. Although MRI examination is more expensive in comparison with computed tomography and ultrasound, this modality is widely used due to higher spatial resolution [25] and absence of ionizing radiation. MRS methods offer an opportunity to assess relative tissue metabolite concentrations based on the chemical shift phenomenon [26] and are very useful in clinical and biomedical studies for examining metabolic changes in vivo non-invasively [25,27]. In particular, 1H MRS is successfully used to determine relative lipid concentration in hepatic tissue [28], whereas 31P MRS allows to detect phosphorylated metabolites, including high-energy phosphates [25]. MRS methods are implemented to investigate the hepatic metabolic state and are applied as non-invasive diagnostic techniques for studying hepatobiliary pathology [29–31]. Despite the fact that MRI and MRS have become increasingly important imaging techniques for investigation of patients with liver and biliary disease [23,24], there are few papers describing application of in vivo MR-techniques for studying liver fluke infection [32–35] and no papers depicting the use of these methods for investigating O. felineus. In this study we used magnetic resonance techniques (MRI/MRS) for differential assessment of liver abnormalities induced by O. felineus in an experimental animal model. We also
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found out correlations between the MR-findings and the histological data as well as the data of the biochemical analysis of liver tissue.
Materials and methods Ethics statement The experimental protocol was approved by the Bioethics Review Committee of the Institute of Cytology and Genetics, Siberian Branch, Russian Academy of Sciences, Novosibirsk (No. 30 from 20.11.2015). All animal experiments were conducted according to the principles of the Guide for the care and use of laboratory animals [36].
Experimental opisthorchiasis model Metacercariae of O. felineus were obtained from naturally infected fish (Leuciscus leuciscus) caught in the river in the endemic areas of Western Siberia (the Tom, Tomsk), Russia. A permit was not required to collect these fish according Federal law of the Russian Federation No. 166 “About fisheries and conservation of water biological resources” from 20.12.2004. For the experiment, 5-week-old male hamsters (n = 8) (Mesocricetus auratus) were infected intragastrically with 50 metacercariae per hamster, according to the previously described protocol [37]. Age-matched intact male hamsters (n = 8) were used as controls. The hamsters were housed two in a cage (OptiRAT) under conventional conditions and were permitted ad libitum access to food and water. The animals were handled in pathogen-free environment. At 8 weeks post-infection, the hamsters were scanned with MRI and MRS in vivo. Following the MR examinations, the subsets of the infected and intact animals were deeply anesthetized with carbon dioxide and euthanized by decapitation. Blood and liver samples were collected for examination from each hamster from the control (n = 8) and infected (n = 8) groups. Serum and blood analyses were performed using routine procedures (Methods in S1 Text).
MRI and MRS in vivo All 1H/31P MR experiments were performed on a horizontal tomographic scanner with magnetic field intensity of 11.7 T (Bruker, Biospec 117/16 USR, Germany). Prior to MR examinations, the animals were fasted overnight. The animals were anaesthetized with gas anesthesia (Isofluran; Baxter Healthcare Corp., Deerfield, IL) using a Univentor 400 Anesthesia Unit (Univentor, Zejtun, Malta). The animal body temperature was maintained with a water circuit installed into the table bed of the tomographic scanner, which maintained the temperature of 30˚C on its surface. A pneumatic respiration sensor (SA Instruments, Stony Brook, NY) was placed under the lower body part, which allowed to control the anesthesia depth. MRI. All MR images were recorded with a receiver—transmitter 1H volume coil (T11440V3). High-resolution T2-weighted images of the hamster liver (section thickness 1 mm; field of vision (FOV) 4.0 × 4.0 mm; matrix 512 × 512 dots) were acquired with respiratory triggering using TurboRARE (Rapid Imaging with Refocused Echoes) method with pulse sequence parameters: TEeff = 18 ms, TR = 900 ms, Flip Angle = 180˚, RARE Factor = 4. Serial images in axial and coronal orientation were recorded for each animal. 1H/31P-MRS. Proton and phosphorus spectra were recorded with a receiver—transmitter double-tuned 1H/31P surface coil (T11619V3). The coil was positioned to the right upper abdominal quadrant of the hamster liver in the right lateral decubitus position, which allowed to minimize the signal capture by muscles and decrease movement artifacts. Before each session, recording of 3 orthogonal packages of liver sections (Multi-slice Tri-Pilot scanning) was conducted, to guide the correct coil positioning relative to liver tissue (in case of infected
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hamster to capture liver damaged zone). The protons spectra were recorded using the spatially localized PRESS (Point Resolved Spectroscopy) method with TE = 20 ms, TR = 2.5 ms, and 256 replicates, voxel size 5×5×5 mm. Water signal in the spectra was suppressed by the variable power and optimized relaxation delays (VAPOR) method. The phosphorus spectra were recorded using the non-spatially localized single-pulse method with TE = 3 ms, TR = 100 ms, and 2,048 replicates. The spectrum range was 40 ppm, which made it possible to obtain information about the tissue contents of 5 substances represented by 7 peaks, phosphomonoester (PME; 6 ppm), inorganic phosphate (Pi; 5 ppm), phosphodiester (PDE; 2 ppm), creatine phosphate (PCr; 0 ppm originated from muscle contamination), and nucleoside triphosphate (NTP; 2.7, 7.8, and 16.5 ppm). MRS data processing. Time domain data were Fourier transformed after Gaussian multiplication (center, 0 ms; width, 30 ms) and phase corrected. Quantification of the spectral peak areas was performed using the TOPSPIN 2.0 PV software package (Brucker), including polynomial baseline correction followed by frequency domain curve peak integration. The 1H and 31P metabolite concentrations were calculated from the peak areas and expressed relative to an area under the curve. Intracellular pH was calculated based on the chemical shift difference δ between Pi and α-NTP (δ = fPi-fαNTP-7.56) using the Henderson-Hasselbalch equation [38]: pHi ¼ 6:75 þ log10½ðdPi
3:27Þ=ð5:69
dPiÞ
ð1Þ
Histological analysis Liver samples (from posterior segments of the right lobe) were fixed in 10% buffered formalin, and embedded in paraffin. Tissue sections were cut into 4–5 μm-thick slices and stained with hematoxylin and eosin. Histological analysis was performed with the optical microscope Axiostar plus (Carl Zeiss, Germany). For differential staining of collagen in the liver samples, Van Gieson’s method was used. Fibrosis was graded according to METAVIR score [39]. Hepatic steatosis was graded on a 0–3 scale through visual estimation of the percentage of hepatocytes containing intracellular vacuoles of fat [40].
Liver tissue analysis Two samples of liver tissue were taken from posterior segments of the right lobe and medial segments of the left lobe for each animal. The samples were divided into five pieces in accordance with subsequent examinations and immediately placed in liquid nitrogen. Homogenization was performed using Tissue Grinders (PELLET PESTLE1 Cordless Motor, Kimble Chase, TN) on ice. Lipids assay. Extraction of lipids from 200 mg of liver tissue (two pieces for each animal) was performed using Folch method [41]. The concentrations of cholesterol, triglycerides and phospholipids in the lipid extract were determined spectrophotometrically (SF2000, Russia) using Chronolab kits (Spain). The total lipids were gravimetrically determined by drying 2 ml of liver lipid extract in a glass vial. ATP assay. 60 mg of liver tissue (two pieces for each animal) was homogenized in 1 ml of 3% trichloroacetic acid and centrifuged at 4,000 g, 4˚C, 20 min. The pH of the supernatant was adjusted to 7.4 with 1 M Tris. The ATP concentration was measured on Anthos lucy2 (Asys Hitech GmbH, Austria) using the Adenosine 50 -triphosphate (ATP) Bioluminescent Assay Kit (Sigma-Aldrich, St. Louis, MO). Protein assay. 30 mg of liver tissue (two pieces for each animal) was homogenized in 400 μl of NP-40 buffer (150 mM sodium chloride, 1.0% Triton X-100, 50 mM Tris, pH 8.0) and centrifuged at 12,000 g, 4˚C, 20 min. Then the protein concentration was determined in
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the supernatant using Bradford assay [42]. Bovine serum albumin (BSA fraction V, Sigma) was used as a protein standard. Glycogen assay. 20 mg of liver tissue (two pieces for each animal) was homogenized in 1 ml of 0.3 M HClO4. The homogenate was incubated with aminoglucosidase (AG, Sigma) in 50 mM sodium acetate, 0.02% BSA, pH 5.5 for 2 h at RT, and centrifuged at 10,000 g for 1 min. The amount of the released glucose was determined by the Hexokinase Method [43] using the Glucose Reagent (Vector-Best, Russia). Glucose (Sigma) was used as a standard. Free glucose in the homogenate was measured without addition of AG and subtracted from AG-treated samples. Western blot. 20 mg of liver tissue was homogenized on ice using dounce homogenizer in 1 ml of lysis buffer (50 mM Tris-HCl (pH 7.5), 250 mM sucrose, 5 mM sodium pyrophosphate (NaPPi), 50 mM NaF, 1 mM EDTA, 1 mM dithiothreitol (DTT), 0.5 mM phenylmethylsulfonyl fluoride (PMSF)) supplemented with Mammalian Protease inhibitor Cocktail (P8340 Sigma). The protein concentration was quantified by Bradford assay, using BSA (Sigma) as a standard. Following lysis sodium dodecyl sulfate (SDS) was added to a final concentration of 0.2% and samples are boiled for 5 min. The samples (twenty micrograms of total proteins) were loaded onto a 15% SDS-PAGE gel according to Laemmli protocol [44]. The separated proteins were transferred to nitrocellulose membranes. The membranes were blocked with 3% non-fat milk in Tris-buffered saline (TBS) with 0.1% Tween 20 and 50 mM NaF for 1 h at room temperature and probed overnight at 4˚C with primary antibodies in 5% BSA supplemented with 50 mM NaF, followed by incubation with Goat Anti-Rabbit IgG H&L (HRP) (ab6721) (Abcam, USA) in TBS supplemented with 5% milk and 50 mM NaF for 1 h at room temperature. The membranes were developed with a ECL Chemiluminescent Substrate Reagent Kit (Thermo Scientific, USA) and scanned by G:box XT4 (Syngene). The band intensities were quantified using GeneTools (Synegene). The primary antibodies Phospho-AMPKα (Thr172) (CellSignaling technology, Inc.) and Anti-beta Actin antibody (ab8227) (Abcam, USA) were used.
Statistical analysis Statistical analyses were performed using IBM SPSS Statistics for Windows, Version 21.0 (Armonk, NY: IBM Corp.). Data were presented as median (range) for data with non-normal distribution and mean (SD) for data with normal distribution. The data were tested by the Shapiro—Wilk test for normality. The differences among continuous variables with normal distribution were analyzed by the t-test and among continuous variables with non-normal distribution—by the Mann-Whitney test. For correlation analyses, the Person (for continuous variables) and Spearman (for ordinal variables) correlations were used. The P value below 0.05 was considered as significant.
Results Histological analysis Histological analysis data showed that O. felineus infection resulted in derangement of the liver architecture, whereas the trabecular pattern mostly remained intact. Hepatocytes had various sizes and patchy exhibited cloudy swelling. Cholangitis in the portal tract was accompanied by pronounced periductal and mild portal fibrosis as well as focal cystic dilatations of the intrahepatic bile ducts. Small bile ductular proliferation and irregular periportal (mostly moderate) infiltration of inflammatory cells, with predominant population of lymphocytes and histiocytes, occurred (Fig 1). Fibrosis was irregular; patchy, incomplete, thin fibrous septa (portal to portal and portal to central bridge) were observed (Fig 1A, 1B and 1C). In whole, chronic
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Fig 1. Histological analysis of hamster liver. (A) Microphotographs of Opisthorchis felineus infected hamster liver at 8 weeks post-infection. An adult fluke is found in bile duct lumen. Small bile duct proliferation and chronic cholangitis accompanied by inflammatory infiltration in the bile duct wall; hematoxylin-eosin staining, ×100. (B) Mild portal fibrosis with thin septa formation (portal to portal and portal to central) and (C) pronounced periductal fibrosis with chronic inflammation around the bile duct are observed. In adjacent hepatic tissue, fibrosis with porto-portal septa formation takes place. Van Gieson’s staining showing extensive deposition of mature collagen fibers, ×100. (D) Microphotographs of the uninfected hamster liver. No pathological changes are observed. Hematoxylin-eosin staining, ×100 and (E) Van Gieson’s staining, ×100. https://doi.org/10.1371/journal.pntd.0005778.g001
cholangitis (mild to moderate inflammatory activity, A1-A2 according to METAVIR score) and mild chronic hepatitis were diagnosed, and the stage of fibrosis varied from 1 to 2 according to METAVIR score. There were no lipid droplets in the tested liver samples. No pathological changes took place in the liver in the reference group (Fig 1D and 1E).
Blood and serum analysis The results of blood and serum analyses are given in Results in S1 Text and Table in S1 Table. In the infected group of hamsters the levels of alanine aminotransferase (ALT) and gammaglutamyl transferase (GGT) increased markedly. Significant elevation of cholesterol, triglycerides, and low-density lipoproteins (LDL) in serum of the infected animals was detected. All the above listed serum parameters correlated with the fibrosis stage. The concentration of albumin in serum of the infected hamsters was lower than in the reference group and was accompanied by a statistically significant increase in the urea concentration. However, there was no correlation between these parameters (r = -0.271, p = 0.309).
Biochemical analysis of liver tissue The liver to body index increased in the infected animals, while the spleen to body index remained unchanged (S1 Table). The results of liver tissue biochemical analysis are given in Table 1. There was a statistically significant rise in the cholesterol level in opisthorchiasis, and the cholesterol to phospholipid ratio also elevated in the infected liver. Total lipid content, triglycerides and phospholipids as well as glycogen did not differ between the control and infected groups of hamsters. A significant decrease in the protein concentration occurred in the infected livers. However, the calculated total protein content in the entire liver did not differ between both control and experimental groups due to enlargement of the organ in the infected hamsters. It is important to note, that infection did not lead to lowering of the ATP concentration in liver tissue.
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Table 1. Liver tissue biochemical analysis. Metabolite#
Control
Protein, mg g-1 Total cholesterol, mg g
-1
Infected
Correlation with fibrosis stage
mean
SD
mean
SD
r
p-level
120.80
17.19
94.92*
15.62
-0.720
0.002
1.80
0.25
2.64*
0.59
0.781
0.000
Triglycerides, mg g-1
2.84
0.39
3.29
1.23
0.282
0.290
Phospholipids, mg g-1
9.89
0.84
8.93
1.05
-0.447
0.082
Total lipids, mg g-1
42.77
4.91
39.88
4.38
-0.136
0.615
Triglycerides to phospholipid ratio
0.26
0.04
0.32
0.09
0.463
0.071
Cholesterol to phospholipid ratio
0.37
0.06
0.60*
0.10
0.885
0.000
ATP, nmol g-1
2.77
0.66
2.94
1.04
-0.032
0.905
Glycogen, mg g-1
4.17
3.41
6.11
2.16
0.152
0.573
#
Concentration calculated per gram wet weight of tissue
*difference between the infected and the control group is significant at p
